Ras mutations are a hallmark of human tumors, including those of the thyroid gland. Ras mutations are found at high frequency in follicular adenomas and carcinomas. Although Ras mutations are infrequent in papillary carcinomas, ret mutations occur at high frequency in these tumors and RET has been shown to signal through Ras. Despite years of active investigation, the contribution of Ras to neoplastic transformation is not well understood. Ras signals through complex signaling networks that are utilized in a cell type-dependent manner. The phenotypic consequences that follow Ras activation depend upon the level and duration of Ras activity, the effectors activated by Ras and importantly, cell context. The effects of activated Ras in primary thyroid cells are unusual. Unlike primary fibroblasts where activated Ras induces growth arrest, Ras stimulates sustained proliferation in primary human thyroid cells. We have shown that acute expression of activated Ras stimulates apoptosis in rat thyroid cells. In these cells as in human thyrocytes, apoptosis is preceded by cell proliferation. However, cell cycle progression in response to acute Ras expression is highly aberrant. Ras-expressing cells progress through G1, are delayed in S phase and perish by apoptosis. Moreover, the effects of Ras on the cell cycle machinery are strikingly different from those of thyroid cell mitogens. Following its acute expression, Ras decreased cyclin D1 and p27 protein levels, and increased p21 expression. Intriguingly, Ras elicited a marked increase in cyclin-dependent kinase-2 (cdk-2) activity predominantly in apoptotic cells. This was accompanied by the cleavage of cyclin A and p27 selectively in apoptotic cells. As observed in thyroid tumors, cyclin D1 was upregulated in thyroid cells selected to survive constitutive expression of activated Ras. It is our hypothesis that apoptosis induced by Ras is a direct consequence of unrestrained cdk-2 activity initiated by Ras effects on the cyclin-dependent kinase inhibitors, p27 and p21. The specific goals of this application are to determine whether cdk-2 activity is both necessary and sufficient for apoptosis stimulated by Ras, to elucidate the molecular mechanisms that contribute to unrestrained cdk-2 activity and apoptosis following acute expression of activated Ras, and to identify the secondary changes that transpire to allow for the survival of thyroid cells harboring Ras.